1. Field of the Invention
The present invention relates to methods for the cryopreservation of plant cells and to methods for the recovery of plants cells which have been cryopreserved. The invention also relates to plants, viable plant cells and plant cells cultures which have been successfully recovered from cryopreservation.
2. Description of the Background
Cryopreservation is based on the reduction and subsequent arrest of metabolic functions of biological material stored in ultra-low temperatures. Cryogenic preservation of plants and plant cells for extended periods without genetic change and the subsequent recovery of normal plant cells with unaltered characteristics and biosynthetic ability has important implications in plant breeding, biomedical research and genetic engineering. At the temperature of liquid nitrogen (xe2x88x92196xc2x0 C.) almost all metabolic activities the cell ceases and cells can be maintained in this suspended, but viable state for extended periods.
Plant cells are cryopreserved to avoid loss by contamination, to minimize genetic change in continuous cell lines, and to avoid aging and transformation in finite cell lines. Traditional methods for preservation of a desirable plant characteristic involve establishment of colonies of plants in the field because many plants do not breed true from seeds. These field plant depositories demand large inputs of labor and land and incur high risks of loss due to weather, disease or other hazards. An alternative to field colony is the establishment of an in vitro collection of plant tissue under normal or limited growth conditions. For long-term storage, elimination of routine subculturing is desirable because of concerns with mutation, contamination, labor cost and risk of human error associated with tissue culture.
Most biological materials, including plants, cannot survive freezing and thawing from cryogenic temperatures without cryoprotective agents and procedures. A number of cryopreservatives possess properties which can protect a cell from the damaging effects of cryogenic freezing. The essence of cryopreservation is to effect cell dehydration and concentration of the cytosol in a controlled and minimally injurious manner so that ice crystallization in the cytosol is precluded or minimized during, for example, quenching in liquid nitrogen.
In conventional cryopreservation procedures, cell dehydration is effected by freeze-induced concentration of the suspending medium. Deleterious effects of dehydration are mitigated by the presence of cryoprotective agents. Specimens such as cells and organs are equilibrated in a solution containing a cryopreservation agent such as dimethylsulfoxide (DMSO) or ethylene glycol. The suspension is cooled and seeded with an ice crystal at a temperature slightly below its freezing point. The suspension is cooled again at an optimum rate to an intermediate sub-zero temperature such as between about xe2x88x9230xc2x0 C. to about xe2x88x9240xc2x0 C. and finally quenched in liquid nitrogen.
While routine cryogenic preservation of microorganisms, zygotes and animals derived from zygotes is possible, the cryopreservation of plant cells is far from routine and often, different protocols for individual species of plants are necessary.
Tarus trees produces taxol, is a diterpenoid alkaloid originally isolated from the bark of the Pacific yew, Taxus brevifolia (M. C. Wani et al., J. Am. Chem. Soc. 93:2325-27, 1971). Experiments have demonstrated that this compound effectively inhibits the polymerization of microtubles of mammalian cells without undue toxicity and, as such, is an effective anti-tumorigenic agent. Clinical trails identified taxol as extremely effective against refractory ovarian, breast and other cancers. As such, taxol is a breakthrough in chemotherapy because of its rather unique, but basic mechanism of action being fundamental distinct from conventional chemotherapeutic agents (L. A. Rowinsky et al., J. Natl. Cancer Instit. 82:1247-59, 1990).
The most daunting variable in the taxol equation so far is supply. It takes three to six, 100 year old Pacific yews to treat one patient because average yields of taxol are low (Witherup et al., 1990). The production of an amount of taxol needed for treatment and testing will require the destruction of tens of thousands of yews. The yew population has been rendered nearly extinct by logging and as the number of Pacific yews dwindles, medical research must look for other forms of supply for taxol. The usefulness of taxol, as well as many other compounds which may be propagated or harvested in plant cells, has fueled an interest in culturing taxus and other plant cells.
The culturing of plant cells for their biosynthetic ability poses special problems for current technology. Prolonged culturing of plant cells often results in a loss of biosynthetic ability which had been present in the original isolates (Dhoot et al., Ann. Bot. 41:943-49, 1977; Barz et. al., Ber. Dtsch. Bot. Ges. 94:1-26, 1981). Phenotypic alterations also arise which further complicate cell culturing. A protocol for freezing plant cells, especially taxus cells, is an important step in the development of biosynthetic methods for production of useful plant alkaloids such as taxol.
The present invention overcomes the problems and disadvantages associated with current strategies and designs and provides novel methods for cryopreservation and for the recovery of viable cryopreserved plant cells.
One embodiment of the invention is directed to methods for the cryopreservation of plant cells. Plant cells, which may be gymnosperms or angiosperms, are pretreated with a cryoprotective agent and a stabilizer, and acclimated to a reduced temperature. Acclimated cells are loaded with a loading agent which may be the same as the vitrifying agent and the loaded cells vitrified with a vitrification solution. Vitrified plant cells are frozen at cryopreservation temperatures, such as, between about xe2x88x9270xc2x0 C. to about xe2x88x92200xc2x0 C. or less.
Another embodiment of the invention is directed to methods for cryopreserving plant cells. Plant cells to be cryopreserved are pretreated with a cryoprotective agent and a stabilizer, and acclimated to a reduced temperature. Acclimated plant cells are vitrified and frozen at a cryopreservation temperature.
Another embodiment of the invention is directed to methods for cryopreserving plant cells. Plant cells to be cryopreserved are cultured in media comprising a vitrifying agent and a stabilizer at a reduced temperature for a first period of time. The cultured plant cells are further cultured in media containing an increased concentration of the vitrifying agent for a second period of time. Plant cells vitrified in the higher concentration of vitrifying agent are frozen at a cryopreservation temperature.
Another embodiment of the invention is directed to methods for cryopreserving plant cells. Plant cells to be cryopreserved are lyophilized by vacuum evaporation and vitrified in a vitrifying solution. Lyophilization removes about 60% of the water from the cells and in combination with vitrification can remove up to about 95%. The vitrified and lyophilized plant cells are frozen and stored at a cryopreservation temperature by, for example, quenching the cells into liquid nitrogen.
Another embodiment of the invention is directed to methods for recovering plant cells from cryopreservation. Plant cells are cryopreserved according to the methods of the invention. Thawed plant cells are warmed to a temperature above freezing and incubated in a media comprising a cryoprotective agent and a stabilizer. The osmotic agent is removed and viable plant cells recovered.
Another embodiment of the invention is directed to methods for recovering cryopreserved plant cells from cryopreservation. Cryopreserved plant cells are thawed to a temperature above freezing and incubated in media comprising an cryoprotective agent and a stabilizer. The cryoprotective agent is removed such as by dilution of the mixture or pelleting of the cells and viable plant cells recovered.
Another embodiment of the invention is directed to viable plant cells which have been cryopreserved by the method of the invention. Cryopreserved plant cells are not significantly genetically or phenotypically altered by cryopreservation.
Another embodiment of the invention is directed to methods for recovering cryopreserved plant cells in suspension. Cryopreserved plant cells are thawed to a temperature above freezing. Thawed plant cells are incubated in liquid suspension and viable cells recovered in liquid media without a need for solid or semi-solid culture.
Another embodiment of the invention is directed to viable plants and plant cells cryopreserved and to viable plants and plant cells recovered by the methods of the invention. Cells are not significantly genotypically or phenotypically altered by the cryopreservation process and have a high proportion of survival.
Other embodiments and advantages of the invention are set forth, in part, in the description which follows and, also in part, will be obvious from this description or may be learned from the practice of the invention.